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Biocompatible Polymer–Peptide Hybrid-Based DNA Nanoparticles for Gene Delivery | Izon Science
Page Count: -. Page Size: -. For more information samaneh. As elasticity is one of the main mechanical properties of cardiovascular tissues, it is important to replicate the elasticity of native tissues using elastomeric biomaterials to create functional implants. The synthetic polymeric scaffolds developed so far show plastic deformation under high strain amounts and therefore fail to replicate the elasticity of innate tissues. To address these limitations, we attempt to develop a synthetic mimic of elastin, as the most important protein that provides the tissue with elasticity, using two different strategies.
New silicon peptide biopolymers
The first strategy is a block-copolymer structure in which Elastin-like polypeptide ELP moieties will be alternated with a hydrophilic domain; this structure closely resembles the structure of native elastin. Bacterial killing was evaluated by measuring intracellular ATP levels, an energy parameter commonly used as an indicator of cell injury and viability .
The 72 hr biofilm formed on the bottom of the wells was scraped using a pipette tip and collected into a set of 15 ml tubes. The total biofilm yield was assessed using crystal violet staining as follows. The acetic acid was transferred to wells of a new well microtiter plate that was placed in a microplate reader and absorbance OD nm was measured.
Confocal laser scanning microscopy CLSM was used to explore the vitality of bacteria in the different depth layers of the biofilm.
Briefly, wells were washed, incubated for 15 min in a solution containing propidium iodide and SYTO 9 and washed again. To read the results directly, the wells were coated with emulsion oil to prevent dehydration. The biofilm was quantified by measuring the area occupied by the bacteria with the aid of Image Pro 4. The presented data are the mean and standard deviation of triplicates of a representative experiment repeated three times. Growth of E. Percent growth inhibition was calculated compared with that of untreated bacteria during the logarithmic phase of the non treated bacteria.
Elastin mimetic polymer-peptide hybrids
Generation time was calculated from each curve using the section representing the exponential growth phase C. Release of the CKGGK lipopeptide from two biodegradable polymers was monitored over one week in two modes. In the first, the antibacterial action of CKGGK released into the medium that came in contact with the formulation every 24 hrs was measured see Fig.
In the second, the bacteria were added to the wells with CKGGK that was released from the polymer and accumulated for one week see Fig. The anti -E.
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The final optical density of the treated bacteria, which was lower than that of the untreated ones. The slope of the curve generation time, see Fig. Fresh medium was added to the first line of wells and was transferred every 24 hrs to a new line below for a week. Percent growth inhibition calculated compared with that of the non- treated bacteria during the logarithmic phase of the non treated bacteria. Generation time was calculated from each curve using the section representing the exponential growth phase C, E.
The vehicle formulation itself does not possess anti-biofilm activity. The optical density of the polymers alone without the bacteria was subtracted from the results of the biofilm that came in contact with the formulation and the polymer.
The level of ATP indicates the active metabolism of a cell. ATP levels in E. These findings led to the question whether the luminescence values are derived from bacterial number, the metabolic status or both. In addition, a similar experiment was performed in which the biofilm was first grown for 48 hrs and then the tested materials were added to verify if CKGGK can affect an already constructed biofilm. The results were similar to those above where the materials were added immediately after inoculating the bacteria. This may indicate that the formulation and the peptide each have an anti-metabolic effect even after the biofilm is formed.
Biofilm was exposed to the formulation for 72 hrs and ATP was measured as described in Materials and Methods. Ef represents the untreated bacteria as control, KGGK - bacteria treated only with peptide ; formulation - bacteria treated with sustained release peptide; polymer - bacteria treated only with polymer as control. The P SA:RA polymer had a strong inhibitory activity against biofilm formation as seen by the reduction in bacterial load.
The medium was discarded and the wells were washed gently with PBS. The live bacteria were stained with green dye, the dead bacteria were stained with a red dye. Images were taken using an Olympus confocal microscope [A, B]. The black column represents the dead bacteria, the white column represents the live bacteria. Their nonspecific mode of action, which is based on physical membrane disruption, is effective against various bacteria and is less likely to induce bacterial resistance than antibiotics. Recently, synthetic AMPs mimicking these strategic antibacterial agents have been gaining interest.
Combining sustained release and an antimicrobial compound holds many advantages and has proved itself in the past. In this study a potent antimicrobial agent was identified against E. The antibacterial effect in this study was tested against E. We chose this bacterium as an example of a pathogen that causes severe nosocomial infections and as an example of a strongly forming biofilm bacterium.
Interestingly, root canal treated teeth are about nine times more likely to harbor E. This frustrating rate of post treatment disease is mainly attributed to the limitations of the present technology that offers no tool to combat intra-canal infection following the cleaning and shaping stage of the endodontic treatment . The tested antibacterial peptides were first assayed in suspension against planktonic E. This may be due to the differences in E. As hBD3 is a costly peptide, high concentrations are predestined to be irrelevant as a conventional therapeutic agent and thus were not tested further.
Interestingly, in some experiments at low concentrations bacterial growth was not inhibited but rather accelerated. The exact mechanism of this opposite outcome is unknown, but the main assumption is that somehow the bacteria overcome lower concentrations of the lipopeptide and show accelerated growth compared with the untreated bacteria.
This phenomenon needs to be considered when dealing with the amount of peptides that are released from the polymer. Indeed calculation of bacterial number using a calibration curve revealed that the final bacterial load was lower by one order of magnitude in the treated wells. Additionally, the slope of the curve representing the bacterial growth rate generation time was more moderate in the treated bacteria, showing that the peptide is released into the medium. The generation time of the bacteria treated with each of the formulations and especially with P SA:RA was longer compared with that of the non-treated bacteria.
Bacteria grow naturally as biofilm, especially E. Moreover, E. Therefore, in the second part of the study the anti-biofilm effect was tested. In the first, crystal violet was used to stain and measure biofilm mass. In the second, an ATP bioluminescence assay was performed and used as a viability indicator. All three experiments revealed inhibition of biofilm formation when E.
The three aspects examined were the amount of biofilm, its metabolic state and bacterial viability. Interestingly, the formulations were effective against a biofilm in the process of formation and against an established biofilm mature biofilm. This is an important finding considering the fact that mature biofilm is much harder to treat because of its virulence factors.
It can be suggested that a formulation that was shown to be active against ATCC v is likely to be potent against other E. The polymer candidates which contain fatty acids have several advantages over other biodegradable polymers such as: flexibility, low melting point, improved handling and provide better degradation and release profiles . As previously reported, biodegradable polyanhydrides and polyesters are useful materials for controlled drug delivery. Fatty acids are suitable candidates for the preparation of biodegradable polymers, as they are natural body components and hydrophobic, and thus may retain an encapsulated drug for longer time periods when used as drug carriers .
Moreover, it was shown that these polymers are biocompatible . As described before, this polymer-peptide interaction may hold many advantages over the peptide by itself, such as improved solubility, reduced immunogenicity, increased stability against degradation and prolonged biological activity .